In situ analysis of tissues

ABSTRACT

The present invention provides for the simultaneous assessment of a plurality of tissue regions or microregions, the benefit being homogeneity of the sampling, both in terms of tissue content and timing. Discrete regions of a tissue sample, such as those demarcated by microwells formed within the tissue itself or tissue plugs removed from the tissue in a spatially referenced fashion, can be treated with one or more physical or chemical treatments to liberate target molecules of interest. Subsequent analysis of said target molecules by, e.g., mass spectroscopy, permits identification of a variety of biological parameters, including those associated with disease or therapy.

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 60/653,665, filed Feb. 17, 2005, the entirecontents of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularbiology. More particularly, it concerns measurement of biologicalmolecules in situ in a tissue sample. Specifically, the inventionprovides for creation of a series of microwells in a single tissue ortissue region, thereby permitting analysis of proteins, nucleic acids,lipids, carbohydrates, drugs and other biomolecules in a homogeneous setof reactions.

2. Description of Related Art

With the completion of the Human Genome Project, emphasis is shifting toexamining the protein complement of the human organism. This has givenrise to the science of proteomics, the study of all the proteinsproduced by cell type and organism. At the same time, there has been arevival of interest in proteomics in many prokaryotes and lowereukaryotes as well.

The term proteome refers to all the proteins expressed by a genome, andthus proteomics involves the identification of proteins in the body andthe determination of their role in physiological and pathophysiologicalfunctions. The ˜30,000 genes defined by the Human Genome Projecttranslate into 300,000 to 1 million proteins when alternate splicing andpost-translational modifications are considered. While a genome remainsunchanged to a large extent, the proteins in any particular cell changedramatically as genes are turned on and off in response to theirenvironment.

As a reflection of the dynamic nature of the proteome, some researchersprefer to use the term “functional proteome” to describe all theproteins produced by a specific cell in a single time frame. Ultimately,it is believed that through proteomics, new disease markers and drugtargets can be identified. Proteomics also has much promise in noveldrug discovery via the analysis of clinically relevant molecular events.The future of biotechnology and medicine will be impacted greatly byproteomics, but advances are needed to realize the potential benefits.

With the availability of DNA microarray analysis, permitting theexpression of thousands of genes to be monitored simultaneously, theimportance of the proteome cannot be overstated as it is the proteinswithin the cell that provide structure, produce energy, and allowcommunication, movement and reproduction. Basically, proteins providethe structural and functional framework for cellular life.

However, there are several impediments in the study of proteins that arenot inherent in the study of nucleic acids. Proteins are more difficultto work with than DNA and RNA. Proteins cannot be amplified like DNA,and are therefore less abundant sequences are more difficult to detect.Some proteins are difficult to analyze due to their poor solubility. Andunlike DNA, the protein content of a given cell may vary depending onlocal conditions, even with a single organism or organ.

SUMMARY OF THE INVENTION

Therefore, in accordance with the present invention, there is provided amethod for analyzing protein content in a tissue comprising (a)providing an intact tissue comprising a first spatially discretemicroregion; (b) subjecting the microregion to one or more physical orchemical treatments; and (c) analyzing a protein sample from themicroregion, thereby providing analysis of protein content in thetissue. The intact tissue may further comprise at least a secondspatially discrete microregion, and the method further comprisessubjecting the second spatially discrete microregion to one or morephysical or chemical treatments, and analyzing discrete a protein samplefrom the second spatially discrete microregion, and the protein contentof the first and second spatially discrete microregions may be compared.Protein encompasses both peptides and polypeptides. One or more of thesteps may be automated.

The provision of a microregion may involve different aspects. In oneembodiment, the microregion may comprise a well in a larger tissuesection. The well may be a true depression or hole in the tissue, or achannel structure created in the tissue. Multiple wells, depressions orholes can be created in a spatially distinct manner. Alternatively, theintact tissue may be extracted from a larger tissue section, i.e., atissue plug. The plug may then be transferred to a support for furthertreatment and analysis, and may be placed adjacent to other plugs in aspatially distinct manner to maintain the same or a pre-determinedrelationship to larger tissue section(s) from which they were extracted.

The tissue may be animal tissue, for example, heart tissue, livertissue, kidney tissue, prostate tissue, breast tissue, ovary tissue,uterine tissue, skin tissue, lung tissue, brain tissue, colon tissue,head & neck tissue, pancreatic tissue, muscle tissue or skin or tissuethat contains body fluids or contains traces of such fluids, such asblood, CSF, urine, saliva, mammary fluid. The animal tissue may be isdiseased or injured, such as cancerous, inflamed, infected, congenitallydiseased, functionally compromised (diabetes, neurodegenerative, oratrophy), traumatized or environmentally insulted. The tissue may beplant tissue, such as leaf tissue, stalk tissue, stem tissue, roottissue, or seed tissue. The plant tissue may be diseased or injured,such as tissue that is infected, congenitally diseased, traumatized orenvironmentally insulted.

The one or more physical or chemical treatments may comprise solventtreatment, detergent treatment, lipase treatment, proteolysis, reactiveagent treatment or labeling. Labeling may comprise treatment with anisotope dilution reagent, a labeled antibody, or an enzyme. Theanalyzing may comprise secondary ion mass spectrometry, laser desorptionmass spectrometry or matrix-assisted laser desorption mass spectrometry,desorption electrospray or electrospray mass spectrometry. Step (c) maybe performed in situ in the microregion, or after removing the proteinsample from the microregion.

The first and second spatially discrete microregions may receivedistinct physical or chemical treatments or the same physical orchemical treatment. The first spatially discrete microregion or tissueadjacent to the first spatially microregion may be subject to a firsttest condition prior to step (b), such as a drug treatment, a nutrienttreatment, a hormone treatment, an enzyme treatment, or a cytokinetreatment. The method may further comprise subjecting the firstspatially discrete microregion or tissue adjacent to a the firstspatially discrete microregion to a second test condition prior to step(b). The second test condition may be different from the first testcondition or the same as the first test condition. The first spatiallydiscrete microregion is subjected to at least a second physical orchemical treatment, which may be distinct from the first treatment.

The microregion may be a microwell in the tissue, such as between 5 and200 microns, between 10 and 100 microns, or about 50 microns. Themicroregion also may be is a tissue cylinder or plug. The method mayfurther comprise generating the microregion, or a plurality ofmicroregions, or a microregion array. The intact tissue may comprise 6microregions, 24 microregions, or 96 microregions.

In accordance with any of the foregoing embodiments, the presentinvention may be modified to examine nucleic acids, lipids,carbohydrates, drugs, metabolites (endogenous and exogenous),xenobiotics, or any other biological molecule.

In another embodiment, there is provided a method for analyzing thedelivery of an exogenous agent to a tissue comprising (a) providing anintact tissue comprising a first spatially discrete microregion; (b)contacting the tissue with the agent; (c) subjecting at the firstspatially discrete microregion to one or more physical or chemicaltreatments; and (d) analyzing a sample from the first spatially discretemicroregion, thereby providing analysis of the delivery of the exogenousagent to the tissue. The exogenous agent may be a peptide or a protein,a nucleic acid, such as an expression construct (e.g., encoding anantisense molecule, a ribozyme, an siRNA, an enzyme, a single-chainantibody, a hormone, a toxin, a tumor suppressor, an inducer ofapoptosis, a cell cycle regulator, a cytokine, or a growth factor), anorganopharmaceutical, or a metabolite.

Analyzing may comprise secondary ion mass spectrometry, laser desorptionmass spectrometry or matrix-assisted laser desorption mass spectrometry,desorption electrospray or electrospray mass spectrometry. The intacttissue may comprises at least a second spatially discrete microregion,and the method further comprises subjecting the second spatiallydiscrete microregion to one or more physical or chemical treatments, andanalyzing a sample from the second spatially discrete microregion. Thesamples from the first and second spatially discrete microregions may becompared. The microregion may be a microwell in the tissue or a tissueplug or cylinder from the tissue. The tissue plug or cylinder may becomprised with an array of tissue plugs or cylinders, each in aspatially discrete relationship to each other.

In yet another embodiment, there is provided a method for analyzing anendogenous metabolite in a tissue comprising (a) providing an intacttissue comprising a first spatially discrete microregion; (b) subjectingthe microregion to one or more physical or chemical treatments; and (c)analyzing a endogenous metabolite sample from the microregion, therebyproviding analysis of endogenous metabolite content in the tissue. Theendogenous metabolite may be an organic acid metabolite, peptidemetabolites, or a part of a sugar.

In another embodiment, there is provided a method for analyzing thedelivery of an exogenous agent to a tissue comprising (a) providing anintact tissue comprising a first spatially discrete microregions; (b)contacting the tissue with the agent; (c) subjecting at the firstspatially discrete microregion to one or more physical or chemicaltreatments; and (d) analyzing a sample from the first spatially discretemicroregion, thereby providing analysis of the delivery of the exogenousagent to the tissue. The exogenous agent may be a peptide or apolypeptide, a nucleic acid, such as an expression construct (e.g.,encoding an antisense molecule, a ribozyme, an siRNA, an enzyme, asingle-chain antibody, a hormone, a toxin, a tumor suppressor, aninducer of apoptosis, a cell cycle regulator, a cytokine, or a growthfactor), an organopharmaceutical, or a metabolite.

Analyzing may comprise secondary ion mass spectrometry, laserdesporption mass spectrometry or matrix-assisted laser desporption massspectrometry, desorption electrospray or electrospray mass spectrometry.The intact tissue may comprise at least a second spatially discretemicroregion, and the method further comprises subjecting the secondspatially discrete microregion to one or more physical or chemicaltreatments, and analyzing a sample from the second spatially discretemicroregion. The content samples from the first and second spatiallydiscrete microregions may be compared. The exogenous agent is deliveredto the tissues as a whole or just to the microregion.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—Image of microwells in rat liver tissue. The image was takenusing an optical microscope.

FIGS. 2A-2B—SEM images of microwells in rat liver tissue. FIG. 2A, lowmagnification image of microwells. FIG. 2B, a magnification of the boxedarea in FIG. 2A.

FIGS. 3A-3C—SEM images of microwells in rat liver tissue with increasingmagnification. FIG. 3A, image with 150 μm magnification. FIG. 3B, imagewith 75 μm magnification. FIG. 3A, image with 6.67 μm magnification.

FIGS. 4A-4C—SEM images taken from different angles. FIG. 4A, SEM imagetaken with a −3 degree tilt. FIG. 4B, SEM image taken with a 0 degreetilt. FIG. 4C, SEM image taken with a +3 degree tilt. FIG. 4D, SEM imagetaken with a +9 degree tilt.

FIG. 5—Microwells in rat liver tissue.

FIG. 6—Microwells in mouse brain tissue.

FIGS. 7A-7B—Images of microwells. FIG. 7A, microwells in mouse embryo.FIG. 7B, microwells in human brain.

FIGS. 8A-8D—Images of microwells in rat kidney. Microwells weregenerated by application of 12 μm rat kidney by 1× (FIG. 8A), 2× (FIG.8B) and 3× (FIG. 8C) 3 DPS 90% acetonitrile, 0.1% TFA spotted. A 3×magnification of the tissue section is shown in FIG. 8D.

FIGS. 9A-9H—Confocal microscopy of microwells in mouse brain. FIG. 9A,an image of the entire mouse brain is shown. FIG. 9B, a magnification ofthe top of well A1 in FIG. 9A is shown. Confocal microscopy of amicrowell is shown at the depths of 3 μm (FIG. 9C), 4 μm (FIG. 9D), 5 μm(FIG. 9E), 6 μm (FIG. 9F), 7 μm (FIG. 9G), and 8 μm (FIG. 9H).

FIGS. 10A-10B—Optical microscopy images. Images of a microwell in ratliver are shown in FIG. 10A and FIG. 10B.

FIGS. 11A-11C—Mass spectroscopy (MS) of microwells generated by trypticdigestion. FIG. 11A, MS data indicating the presence of the sequence ofthe mouse β tubulin protein are indicated with the “*” symbol. FIG. 11B,MS data from fragmentation of the 1619.8 Da peptide. FIG. 11C, MS datafrom fragmentation of the 1052.6 Da peptide.

FIGS. 12A-12B—Mass spectroscopy (MS) of microwells generated by trypticdigestion showing identification of the myelin basic protein. FIG. 12A,MS data indicating the presence of the sequence of the sequence of MBP6,AAA39496, M calc 18476 are indicated with the “*” symbol. FIG. 12B, MSdata from fragmentation of the 1339.7 Da peptide.

FIG. 13—Microwell formation based on one pass with varying dropletnumber. Ethanol washed rat liver (12 μm) is spotted with 1-9 droplets(start/stop mode) of the following solvent: 0.5% acetic acid mixed to asolvent mixture of 30% isopropanol, 10% acetonitrile and 60% water.Droplets deposited A) 1 DPS, B) 2 DPS, C) 3 DPS, D) 4 DPS, E) 5 DPS, F)6 DPS, G) 7 DPS, H) 8 DPS, I) 9 DPS

FIGS. 14A-C—Microwell formation based on multiple pass versus multipledroplets. (FIG. 14A) Magnified microwells, no stain. (FIG. 14B)Magnified microwells, stained. (FIG. 14C) Close up (40×) of methyleneblue stained microwell formed in 12 μm rat liver (solvent was 0.5%acetic acid, 30% isopropanol, 10% acetonitrile, 60% water). For FIGS.14A-B, top row=4 passes, middle row=6 passes, bottom row=10 passes, leftcolumn=3 DPS, middle column=4 DPS, right column=5 DPS.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The PresentInvention

Proteomics is an extremely powerful tool in examining cellular function,and provides a complementary analysis to genomics efforts. While it issomewhat more complicated to examine protein expression profiles, massspectrometry (MS), because of its extreme selectivity and sensitivity,has now become a favored tool in the global examination of proteinexpression. However, a limitation on any analysis of this nature is theneed to interrogate molecular changes in discrete tissue samples whilepermitting high throughput.

Traditional methods of examining proteomes with MS involve homogenizingsmall samples of tissues, using separative techniques such as 2D gels orliquid chromatography, which are followed by MS for detection. Althoughthis method gives adequate results, it is tedious, labor intensive anddestroys any spatial fidelity in the sample due to the homogenizationprocess. Therefore, current approaches to MS quantification of proteinexpression require substantial improvements in sample processing andutilization.

The present inventors have developed a method for analyzing proteinexpression in situ, i.e., directly in intact tissues and within discreteareas thereof. In particular embodiments, micron-sized wells are createdin intact tissues of interest. The wells create “vessels” in whichchemistries can be performed, such as detergent extractions, labelingreactions, etc. Subsequently, the wells can be interrogated with varioustechniques, particular mass spectroscopy.

The present invention is not limited, however, to proteomicsapplications. With relative ease, the methods described herein may beadvantageously applied to examining the lipid, carbohydrate, nucleicacid, metabolite or even drug content of a tissue. The details of theinvention are described in the following pages.

II. Mass Spectrometry

By exploiting the intrinsic properties of mass and charge, massspectrometry (MS) can resolved and confidently identified a wide varietyof complex compounds, including proteins. Traditional quantitative MShas used electrospray ionization (ESI) followed by tandem MS (MS/MS)(Chen et al., 2001; Zhong et al., 2001; Wu et al., 2000) while newerquantitative methods are being developed using matrix assisted laserdesorption/ionization (MALDI) followed by time of flight (TOF) MS(Bucknall et al., 2002; Mirgorodskaya et al., 2000; Gobom et al., 2000).

A. ESI

ESI is a convenient ionization technique developed by Fenn andcolleagues (Fenn et al., 1989) that is used to produce gaseous ions fromhighly polar, mostly nonvolatile biomolecules, including lipids. Thesample is injected as a liquid at low flow rates (1-10 μL/min) through acapillary tube to which a strong electric field is applied. The fieldgenerates additional charges to the liquid at the end of the capillaryand produces a fine spray of highly charged droplets that areelectrostatically attracted to the mass spectrometer inlet. Theevaporation of the solvent from the surface of a droplet as it travelsthrough the desolvation chamber increases its charge densitysubstantially. When this increase exceeds the Rayleigh stability limit,ions are ejected and ready for MS analysis.

A typical conventional ESI source consists of a metal capillary oftypically 0.1-0.3 mm in diameter, with a tip held approximately 0.5 to 5cm (but more usually 1 to 3 cm) away from an electrically groundedcircular interface having at its center the sampling orifice, such asdescribed by Kabarle et al. (1993). A potential difference of between 1to 5 kV (but more typically 2 to 3 kV) is applied to the capillary bypower supply to generate a high electrostatic field (10⁶ to 10⁷ V/m) atthe capillary tip. A sample liquid carrying the analyte to be analyzedby the mass spectrometer, is delivered to tip through an internalpassage from a suitable source (such as from a chromatograph or directlyfrom a sample solution via a liquid flow controller). By applyingpressure to the sample in the capillary, the liquid leaves the capillarytip as a small highly electrically charged droplets and furtherundergoes desolvation and breakdown to form single or multicharged gasphase ions in the form of an ion beam. The ions are then collected bythe grounded (or negatively charged) interface plate and led through anthe orifice into an analyzer of the mass spectrometer. During thisoperation, the voltage applied to the capillary is held constant.Aspects of construction of ESI sources are described, for example, inU.S. Pat. Nos. 5,838,002; 5,788,166; 5,757,994; RE 35,413; 6,756,586,5,572,023 and 5,986,258.

B. ESI/MS/MS

In ESI tandem mass spectroscopy (ESI/MS/MS), one is able tosimultaneously analyze both precursor ions and product ions, therebymonitoring a single precursor product reaction and producing (throughselective reaction monitoring (SRM)) a signal only when the desiredprecursor ion is present. When the internal standard is a stableisotope-labeled version of the analyte, this is known as quantificationby the stable isotope dilution method. This approach has been used toaccurately measure pharmaceuticals (Zweigenbaum et al., 2000;Zweigenbaum et al., 1999) and bioactive peptides (Desiderio et al.,1996; Lovelace et al., 1991). Newer methods are performed on widelyavailable MALDI-TOF instruments, which can resolve a wider mass rangeand have been used to quantify metabolites, peptides, and proteins.Larger molecules such as peptides can be quantified using unlabeledhomologous peptides as long as their chemistry is similar to the analytepeptide (Duncan et al., 1993; Bucknall et al., 2002). Proteinquantification has been achieved by quantifying tryptic peptides(Mirgorodskaya et al., 2000). Complex mixtures such as crude extractscan be analyzed, but in some instances sample clean up is required(Nelson et al., 1994; Gobom et al., 2000). Desporption electrospray is anew associated technique for sample surface analysis.

C. SIMS

Secondary ion mass spectroscopy, or SIMS, is an analytical method thatuses ionized particles emitted from a surface for mass spectroscopy at asensitivity of detection of a few parts per billion. The sample surfaceis bombarded by primary energetic particles, such as electrons, ions(e.g., O, Cs), neutrals or even photons, forcing atomic and molecularparticles to be ejected from the surface, a process called sputtering.Since some of these sputtered particles carry a charge, a massspectrometer can be used to measure their mass and charge. Continuedsputtering permits measuring of the exposed elements as material isremoved. This in turn permits one to construct elemental depth profiles.Although the majority of secondary ionized particles are electrons, itis the secondary ions which are detected and analysis by the massspectrometer in this method.

D. LD-MS and LDLPMS

Laser desorption mass spectroscopy (LD-MS) involves the use of a pulsedlaser, which induces desorption of sample material from a samplesite—effectively, this means vaporization of sample off of the samplesubstrate. This method is usually only used in conjunction with a massspectrometer, and can be performed simultaneously with ionization if oneuses the right laser radiation wavelength.

When coupled with Time-of-Flight (TOF) measurement, LD-MS is referred toas LDLPMS (Laser Desorption Laser Photoionization Mass Spectroscopy).The LDLPMS method of analysis gives instantaneous volatilization of thesample, and this form of sample fragmentation permits rapid analysiswithout any wet extraction chemistry. The LDLPMS instrumentationprovides a profile of the species present while the retention time islow and the sample size is small. In LDLPMS, an impactor strip is loadedinto a vacuum chamber. The pulsed laser is fired upon a certain spot ofthe sample site, and species present are desorbed and ionized by thelaser radiation. This ionization also causes the molecules to break upinto smaller fragment-ions. The positive or negative ions made are thenaccelerated into the flight tube, being detected at the end by amicrochannel plate detector. Signal intensity, or peak height, ismeasured as a function of travel time. The applied voltage and charge ofthe particular ion determines the kinetic energy, and separation offragments are due to different size causing different velocity. Each ionmass will thus have a different flight-time to the detector.

One can either form positive ions or negative ions for analysis.Positive ions are made from regular direct photoionization, but negativeion formation require a higher powered laser and a secondary process togain electrons. Most of the molecules that come off the sample site areneutrals, and thus can attract electrons based on their electronaffinity. The negative ion formation process is less efficient thanforming just positive ions. The sample constituents will also affect theoutlook of a negative ion spectra.

Other advantages with the LDLPMS method include the possibility ofconstructing the system to give a quiet baseline of the spectra becauseone can prevent coevolved neutrals from entering the flight tube byoperating the instrument in a linear mode. Also, in environmentalanalysis, the salts in the air and as deposits will not interfere withthe laser desorption and ionization. This instrumentation also is verysensitive, known to detect trace levels in natural samples without anyprior extraction preparations.

E. MALDI-TOF-MS

Since its inception and commercial availability, the versatility ofMALDI-TOF-MS has been demonstrated convincingly by its extensive use forqualitative analysis. For example, MALDI-TOF-MS has been employed forthe characterization of synthetic polymers (Marie et al., 2000; Wu etal., 1998). peptide and protein analysis (Zaluzec et al., 1995;Roepstorff et al., 2000; Nguyen et al., 1995), DNA and oligonucleotidesequencing (Miketova et al., 1997; Faulstich et al., 1997; Bentzley etal., 1996), and the characterization of recombinant proteins (Kanazawaet al., 1999; Villanueva et al., 1999). Recently, applications ofMALDI-TOF-MS have been extended to include the direct analysis ofbiological tissues and single cell organisms with the aim ofcharacterizing endogenous peptide and protein constituents (Li et al.,2000; Lynn et al., 1999; Stoeckli et al., 2001; Caprioli et al., 1997;Chaurand et al., 1999; Jespersen et al., 1999).

The properties that make MALDI-TOF-MS a popular qualitative tool—itsability to analyze molecules across an extensive mass range, highsensitivity, minimal sample preparation and rapid analysis times—alsomake it a potentially useful quantitative tool. MALDI-TOF-MS alsoenables non-volatile and thermally labile molecules to be analyzed withrelative ease. It is therefore prudent to explore the potential ofMALDI-TOF-MS for quantitative analysis in clinical settings, fortoxicological screenings, as well as for environmental analysis. Inaddition, the application of MALDI-TOF-MS to the quantification ofpeptides and proteins is particularly relevant. The ability to quantifyintact proteins in biological tissue and fluids presents a particularchallenge in the expanding area of proteomics and investigators urgentlyrequire methods to accurately measure the absolute quantity of proteins.While there have been reports of quantitative MALDI-TOF-MS applications,there are many problems inherent to the MALDI ionization process thathave restricted its widespread use (Kazmaier et al., 1998; Horak et al.,2001; Gobom et al., 2000; Wang et al., 2000; Desiderio et al., 2000).These limitations primarily stem from factors such as the sample/matrixheterogeneity, which are believed to contribute to the large variabilityin observed signal intensities for analytes, the limited dynamic rangedue to detector saturation, and difficulties associated with couplingMALDI-TOF-MS to on-line separation techniques such as liquidchromatography. Combined, these factors are thought to compromise theaccuracy, precision, and utility with which quantitative determinationscan be made.

Because of these difficulties, practical examples of quantitativeapplications of MALDI-TOF-MS have been limited. Most of the studies todate have focused on the quantification of low mass analytes, inparticular, alkaloids or active ingredients in agricultural or foodproducts (Wang et al., 1999; Jiang et al., 2000; Wang et al., 2000; Yanget al., 2000; Wittmann et al., 2001), whereas other studies havedemonstrated the potential of MALDI-TOF-MS for the quantification ofbiologically relevant analytes such as neuropeptides, proteins,antibiotics, or various metabolites in biological tissue or fluid(Muddiman et al., 1996; Nelson et al., 1994; Duncan et al., 1993; Gobomet al., 2000; Wu et al., 1997; Mirgorodskaya et al., 2000). In earlierwork it was shown that linear calibration curves could be generated byMALDI-TOF-MS provided that an appropriate internal standard was employed(Duncan et al., 1993). This standard can “correct” for bothsample-to-sample and shot-to-shot variability. Stable isotope labeledinternal standards (isotopomers) give the best result.

With the marked improvement in resolution available on modern commercialinstruments, primarily because of delayed extraction (Bahr et al., 1997;Takach et al., 1997), the opportunity to extend quantitative work toother examples is now possible; not only of low mass analytes, but alsobiopolymers. Of particular interest is the prospect of absolutemulti-component quantification in biological samples (e.g., proteomicsapplications).

The properties of the matrix material used in the MALDI method arecritical. Only a select group of compounds is useful for the selectivedesorption of proteins and polypeptides. A review of all the matrixmaterials available for peptides and proteins shows that there arecertain characteristics the compounds must share to be analyticallyuseful. Despite its importance, very little is known about what makes amatrix material “successful” for MALDI. The few materials that do workwell are used heavily by all MALDI practitioners and new molecules areconstantly being evaluated as potential matrix candidates. With a fewexceptions, most of the matrix materials used are solid organic acids.Liquid matrices have also been investigated, but are not used routinely.

III. Tissue Microregions

A. Obtaining Tissue Specimens

In accordance with the present invention, intact tissue samples areobtained by standard methodologies. The tissue samples must be of asufficient size to permit creation of a plurality of microregions, e.g.,at least 1 micron to several millimeters, including sizes in between,such as 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 250 μm, 275 μm, 300 μm, 400μm, 450 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 2 mm, 3 mm,4 mm and 5 mm.

Biopsy procedures will generally involve the sterility required ofsurgical operations, even though the tissues being sample are fromcadavers or animals that will be sacrificed. For internal tissues,incisions will be made proximal to the tissue of interest, followed byretraction, excision of tissue and surgical closing of the incision.Superficial tissue sites are accessed by simple excision of theavailable tissue.

Because tissue viability is an important aspect of the invention,post-surgical treatment requires that tissues be handled such that (a)the integrity of the tissue is maintained and (b) that the cells withinthe tissue, particularly those in the region(s) where microwells will becreated, are not damaged. Appropriate physiologic buffers are generallyapplied to the tissue, or the tissues are immersed therein. The tissuemay also be cooled to appropriate temperatures for limited periods oftime. Steps should be taken to ensure that apoptosis or other cellulardegredation will not be induced in the tissue specimen.

B. Pretreatment

Pretreatment with of tissues prior to creation microregions may proveadvantageous. On particularly useful pretreatment is an ethanol wash,optionally followed by a storage period of minutes to hours. In tests onseveral tissue types, improved well formation was observed using thisapproach. Also, the delivery of matrix in a solvent comprising 10%acetonitrile, 60% water, 30% isopropanol and 0.5% acetic acid providedimproved results.

C. Microregions

A microregion is an area on the tissue sample that comprise substructureor areas of cellular change or areas of unique interest because of theirmorphology. The size ranges from nanometers to millimeters.

The microregions can be made chemically or physical. For example, ablunt object can be used to tap into the tissue to form a well or wells,or cut into the tissue to create a well or wells, or to create amicroplug or microplugs. Microwells can also be made by laser treatment.Examples of cutting include the use of a capillary tube or an array ofcapillary tubes to create a spatially fixed group of microplugs that canbe transferred to a substrate and, optionally, retained within thecapillary array so as to retain structural integrity of the microplugand to prevent contamination of microplugs with adjacent tissue ortreatment. Finally, the wells may be created using chemical preparatorytreatments described below, i.e., proteases, lipase and the like.

The tissue may be affixed to a support to facilities creation of themicroregions, i.e., to provide a stable foundation. Paraffin is one suchsupport, although artificial surfaces such as glass may be utilized.

D. Automation

The process of well creation may be automated using a “microprinting”device that is capable of physically or chemically creating amicroregion in repeatable fashion. The device will be able to deliverysolvents to multiple locations on a tissue in a predetermined pattern,or to extract tissue “plugs” and transfer them to an appropriatesurface, again retaining a predetermined pattern/relationship traceableto their location in the original tissue. In the former case, usingmultiple drops of a solvent in a single print pass produces more wellpronounced microwell structures than single drops. Multipass prints,though also successful when using multiple drops, create larger wells.

IV. Sample Preparation

Once one has obtained and prepared tissues sections containingmicrowells according to the present invention, it will be necessary totreat the microwells in order to liberate proteins for further analysis.A wide variety of techniques may be applied to the microwells includingdetergent extraction, treatment with various enzymes (lipases,collagenases, proteases, nucleases) or even with enzyme inhibitors(protease inhibitors). Examples of these treatments are provided below.

A. Detergent Extraction

In order to perform mass spectroscopic or other analysis of proteinmaterials from samples of the present invention, certain treatments arerequired to prepare the proteins. At a minimum, proteins will besolubilized using detergent extraction. A variety of detergents areavailable for protein extraction, including anionic, cationic,zwitterionic and non-ionic detergents. By virtue of their amphipathicnature, detergents are able to disrupt bipolar membranes to first freeand then solubilize proteins bound in the membrane or found inside thetarget cells.

In selecting a detergent, consideration will be given to the nature ofthe target protein(s), and the fact that anionic and cationic detergentsare likely to have a greater effect on protein structure thanzwitterionic or non-ionic detergents. However, non-ionic detergents tendto interfere with charge-bases analyses like mass spectroscopy, and arealso susceptible to pH and ionic strength. Zwitterionic detergentsprovide intermediate properties that, in some respects, are superior tothe other three detergent types. Offering the low-denaturing andnet-zero charge characteristics of non-ionic detergents, zwitterionicsalso efficiently disrupt protein aggregation without the accompanyingdrawbacks. Exemplary anionic detergents include chenodeoxycholic acid,N-lauroylsarconsine sodium salt, lithium dodecyl sulfate,1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodiumdeoxycholate, sodium dodecyl sulfate and glycodeoxycholic acid sodiumsalt. Cationic detergents include cetylpyridinium chloride monohydrateand hexadecyltrimethylammonium bromide. Zwitterionic detergents includeCHAPS, CHAPSO, SB3-10 and SB3-12. Non-ionic detergents may be selectedfrom N-decanoyl-N-methylglucamine, digitonin, n-dodecyl β-D-maltoside,octyl α-D-glucopyranoside, Triton X-100, Triton X-114, Tween 20 andTween 80.

The present inventors have described a method utilizing a novelcleavable detergent,3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS). Thisdetergent can be used to extract protein contained within the interiorof the cell by disrupting cell membranes. Once the proteins are freefrom the cell, PPS also assists in protein solubilization by shieldingthe hydrophobic regions of the newly extracted protein from unfavorableinteractions with water. The added advantage of PPS over conventionaldetergents such as sodium dodecyl sulfate or n-octylglucoside is thatthe detergent properties that interfere with MALDI mass spectrometry canbe eliminated prior to analysis. PPS was found to improve sensitivity inMALDI analyses of both soluble proteins and membrane proteins withoutdegrading spectral quality. The virtues of this strategy were applied towhole cell extracts (Norris et al., 2003).

B. Lipases

To the extent that lipid removal by detergent extraction is incomplete,one my choose to utilize enzymes to further degrade lipid contaminants.Such enzymes are called lipases, almost all of which exhibit thecatalytic triad Ser-Asp-His (an exception being geotrichium candidumwhich has Ser-Glu-His). The areas of the protein predicted to beinvolved in interfacial activation and conformational change showvarying sequences, but a large number possess some sort of flap coveringthe active site.

Lipases are also characterized by the phenomena of interfacialactivation. At very low substrate concentrations in aqueous solution,the enzymes are inactive. When the substrate concentration is highenough to form lipid micelles, the enzyme becomes activated. Though themechanism in not yet fully understood, it appears that the lipase existsin two (or perhaps more) possible conformations. In one extreme, thereis a conformation in which a structural element covers the active site,while at the other extreme, there is a structure in which the activesite is exposed, allowing substrate to gain access. The micelle mayinitiate exposure of the active site.

Lipases have been isolated from a wide variety of mammalian andmicrobial sources. The mammalian lipases can be split into four groups,the hepatic lingual, gastric and pancreatic lipase and microbial lipasesinto bacterial and fungal. Very little homology has been found withinthe known sequences, the most conserved feature being the consensussequence G×S×G found in the substrate binding site. The above-mentionedcatalytic triad (Ser-Asp-His) is also a highly conserved. However, thisis common to all esterases, not just lipases, as is the α/βhydrolasefold.

Known lipases include triacylglycerol lipase (triglyceride lipase;tributyrase) phospholipase A2 (phosphatidylcholine 2-acylhydrolase,lecithinase A, phosphatidase, phosphatidolipase) lysophospholipase(lecithinase B, lysolecithinase, phospholipase B) acylglycerol lipase(monoacylglycerol lipase) galactolipase, phospholipase A1, lipoproteinlipase (clearing factor lipase, diglyceride lipase, diacylglycerollipase) dihydrocoumarin lipase, 2-acetyl-1-alkylglycerophosphocholineesterase (1-alkyl-2-acetylglycerophosphocholine esterase,platelet-activating factor acetylhydrolase, PAF acetylhydrolase, PAF2-acylhydrolase, LDL-associated phospholipase A2 LDL-PLA(2)),phosphatidylinositol deacylase (phosphatidylinositol phospholipase A2)phospholipase C (lipophosphodiesterase I, Lecithinase C, Clostridiumwelchii α-toxin, Clostridium oedematiens β- and γ-toxins) phospholipaseD, (lipophosphodiesterase II, lecithinase D, choline phosphatase),phosphoinositide phospholipase C (triphosphoinositide phosphodiesterase,phosphoinositidase C, 1-phosphatidylinositol-4,5-bisphosphatephosphodiesterase, monophosphatidylinositol phosphodiesterase.phosphatidylinositol phospholipase C, PI-PLC,1-phosphatidyl-D-myo-inositol-4,5-bisphosphateinositoltrisphosphohydrolase), alkylglycerophosphoethanolaminephosphodiesterase. (lysophospholipase D), glycosylphosphatidylinositolphospholipase D (GPI-PLD, glycoprotein phospholipase D,phosphatidylinositol phospholipase D, phosphatidylinositol-specificphospholipase D, phosphatidylinositol-glycan-specific phospholipase D),phosphatidylinositol diacylglycerol-lyase (1-phosphatidylinositolphosphodiesterase, monophosphatidylinositol phosphodiesterase,phosphatidylinositol phospholipase C, 1-phosphatidyl-D-myo-inositolinositolphosphohydrolase (cyclic-phosphate-forming)),glycosylphosphatidylinositol diacylglycerol-lyase((glycosyl)phosphatidylinositol-specific phospholipase C, GPI-PLC,GPI-specific phospholipase C, VSG-lipase, glycosyl inositol phospholipidanchor-hydrolyzing enzyme, glycosylphosphatidylinositol-phospholipase C,glycosylphosphatidylinositol-specific phospholipase C,variant-surface-glycoprotein phospholipase C).

C. Collagenases

Collagen is the most abundant protein in vertebrates, and occurs inalmost very tissue. However, for many applications, is it necessary toremove collagen in order to analyze other proteins in a sample.Moreover, analysis of collagen may be of only limited interest. As aresult, methods for the removal of collagen are regularly employed intissue dissociation.

Collagenases are enzymes that are able to cleave the peptide bonds intriple helical collagen molecules. Collagenases from Clostridiumhistolyticum have been known and studied for decades. Clostridopeptidaseand clostripain activities also are associated with some collagenasepreparations. Collagenase has been shown effective in isolating intactcells from a variety of tissues including bone, cartilage, thyroid,ovary, uterus, skin, endothelium, neuronal, pancreas, heart, liver andtumors.

D. Nucleic Acid Removal

Elimination of nucleic acids from sample prior to analysis can beachieved by chemical or enzymatic means. Chemical removal involvesprecipitation methods that employ polyehtyleneimine (PEI) orstreptomycin sulfate precipitation, followed by centrifugation.

Alternatively, enzymes that specifically degrade DNA and/or RNA may beused to remove these molecules. Benzonase is a genetically engineeredendonuclease from Serratia marcescens. The protein is a dimer of two 30kDa subunits. The enzyme degrades all forms of DNA and RNA, includingsingle-stranded, double-stranded, linear and circular molecules, and iseffective over a wide range of operating conditions. Some sequencespecificity has been identified, with GC-rich regions being preferred.More selective enzymes that degrade DNA (DNases) or RNA (RNases) can beutilized as well.

E. Buffers

Once extracted, buffers will often be utilized to preserve the integrityof protein samples. Buffers are aqueous composed of a weak acid (protondonor) and its conjugate base (proton acceptor). The acid or base ispartially neutralized and shows little pH change in response to theaddition of stronger acids or bases because of the buffers ability to“absorb” hyrogen ions, which determine pH. The most effective pH rangefor a buffer is generally one pH unit and is centered around the pK_(a)of the system.

In choosing an appropriate buffer system, one generally takes intoaccount the following considerations. (1) The pK_(a) of the buffershould be near the desired midpoint pH of the solution. (2) The capacityof the buffer should fall within one to two pH units above or below thedesired pH values. If the pH is expected to drop during the procedure,choose a buffer with a pK_(a) slightly lower than the midpoint pH.Similarly, if the pH is expected to rise, choose a buffer with aslightly elevated pH. (3) The concentration of the buffer should beadjusted to have enough capacity for the experimental system. (4) The pHof the buffer should be checked at the temperature and concentrationwhich will be used in the experimental system. (5) No more than 50% ofthe buffer components should be dissociated or neutralized by ionicconstituents which are generated within or added to the solution. (6)Buffer materials should not absorb light between the wavelengths of240-700 nm.

Useful buffers include ADA (Na salt), BES, ethyl glycinate, glycine,PBS, lithium citrate, PIPPS, potassium phosphate (mono- or dibasic),sodium citrate, sodium phosphate, TAPS, Tris base, Tris-HCl, MES,Bis-Tris, PIPES (Na salt), ACES, MOPES, TES, HEPES (Na salt), HEPPS,Tricine, Bicine, CHES, CAPS, MOPSO, DIPSO, HEPPSO, POPSO, AMPSO andCAPSO.

Particularly useful buffers for mass spectrometry are volatile buffers,including ammonium bicarbonate and ammonium acetate.

F. Protease Inhibitors

In order to prevent proteins from being non-specifically degradedfollowing extraction, it may be necessary to include inhibitors ofproteases, which are enzymes that hydrolyze peptide bonds. Proteases orpeptidases are usually categorized as endopeptidases, which cleaveinternal bonds, or exopeptides, which remove residues from the terminiof protein chains. Alternatively, proteases may be classified by virtueof their target sites, such as serines or cysteines.

The following protease-selective inhibitors are known to be useful inaccordance with the present invention: antipain dihydrochloride (papain,trypsin); calpain inhibitor I (calpain I and II); calpain inhibitor II(calpain II and I); chymostatin (chymotrypsin); hirudin (thrombin);TLCK.HCl (trypsin, bromelain, ficin, papain); TPCK (chymotrypsin,bromelain, ficin, papain); and trypsin inhibitor (trypsin). Otherinhibitors include APMSF, aprotinin, bestatin, leupeptin, pepstatin,PMSF, and TIMP-2.

G. Proteolysis

In other embodiments, it may be desirable to fragment peptides, albeitin a controlled fashion. There are two basic methods for digestingproteins: enzymatic and chemical methods. Enzymatic digestions are morecommon. An ideal digestion cuts only at a specific amino acid, but cutsat all occurrences of that amino acid. The number of digestion sitesshould not produce too many peptides because separation of peptidesbecomes too difficult. On the other can, too few digestions producespeptides too large for certain kinds of analysis.

The most common digestions are with trypsin and lysine specificproteinases, because these enzymes are reliable, specific and produce asuitable number of peptides. The next most common digestion is ataspartate or glutamate using endoproteinase Glu-C or endoproteinaseAsp-N. Chymotrypsin is sometimes used, although it does not have a welldefined specificity. Proteinases of broad specificity may generate manypeptides, and the peptides may be very short. Of the chemical cleavages,cyanogen bromide is the most common. All the chemical digestions areless efficient than a good enzymatic digest. However they do produceonly a few peptides, which can ease any purification problem.

V. Applications

In various aspects of the present invention, tissues may be utilizedwith or without further treatment. For example, by examining theproteome of various tissues, one can identify subjects that have or areat risk of disease, including infections, cancer, autoimmune disorders,diabetes, or virtually any other condition for which protein aberrationsare known. As stated above, the invention may also be applied to theexamination of nucleic acids, lipids, carbohydrates, or metabolites.

Alternatively, the tissue may have been treated with one or more agentsor environmental factors, and the examination may seek to assess theimpact of those agents or factors on the tissue. The agents or factorsmay be candidate substances and applied as part of a screening assay todetermine their suitability at therapeutic agents. Alternatively, theagents or factors may be those found in the environment, and the assaywill determine whether the agents have a positive or negative impact onthe health or viability of the tissue.

Identification of proteins corresponding to predictive MALDI-TOF signalsinvolves two approaches. First, protein extracts from tissue sampleswill be fractionated by HPLC, 1D SDS-PAGE or solution phase isoelectricfocusing and fractions exhibiting the MALDI-TOF MS signals of interestwill be subjected to tryptic digestion and analysis by LC-MS-MS.Peptides and their corresponding proteins of origin are identified fromMS-MS spectra with Sequest, which correlates uninterpreted MS-MS spectrawith theoretical spectra from database sequences (Eng et al., 1994).Confirmation of the protein identities is based on apparent molecularweight of the MS-MS identified proteins compared to pattern-specificsignals detected in the MALDI profiles.

A second identification approach will pair LC-MS-MS analyses with stableisotope tags. Protein extracts from two samples to be compared (e.g.,samples that differ in MALDI proteome patterns) are chemically taggedwith light and heavy (unlabeled vs. deuterium or ¹³C-labeled) reagents,then combined, digested and the tagged peptides are then analyzed byLC-MS-MS. Peptides derived from the two samples are distinguished bypairs of signals in full scan MS separated by the mass difference of thelight and heavy isotope tags. Pairs of signals whose intensities deviatefrom unity represent proteins that were differentially present in theoriginal two samples. MS-MS spectra acquired from these peaks in thesame LC-MS-MS analyses allow unambiguous identification of thedifferentially expressed proteins. The best-known version of thisapproach uses the thiol-reactive ICAT reagents developed by Gygi andAebersold (Gygi et al., 1999), although newer, acid-cleavable reagentsoffer more efficient recovery of tagged peptides and produce higherquality MS-MS spectra for identification (Zhou et al., 2002). N-terminalisotope tagging of tryptic peptides enables identification of proteinsthat differ in posttranslational modifications rather that proteinexpression level per se (Mason and Liebler, 2003).

A. Diagnostics

In one aspect, the present invention involves the use of massspectroscopy to diagnosis or predict conditions or disease states in asubject. Ideally, the use of the present invention permits replicatesampling to ensure accuracy, but also permits testing for multipletargets in descrete but spatially related portions of a tissue. Tissuesamples may be obtained using protocols described elsewhere in thisdocument.

Conditions that may be diagnosed according to the present inventioninclude, but are not limited to, cancer, infection, congenital disease,exposure to toxicity, diabetes. Generally, the protein expression of oneor more protein targets in the tissue sample will be compared in astandard or known expression level, array or distribution.Alternatively, known healthy tissue may be interrogated in parallel toprovide the “normal control” to which the sample is compared.

B. Monitoring

In another embodiment, the present invention permits the monitoring ofdisease development, disease progression, or the effects of a treatmenton a subject. Such an assay will comprise, essentially, the same steps adiagnostic method with the exception that the timing of the examinationwill be based on (a) a previous negative diagnostic result, (b) aprevious positive diagnostic result, or (c) a prior treatmentapplication.

C. Screening

In another aspect, the present invention comprises methods for screeningdrugs for the ability to modulate protein content of a cell. Theseassays may comprise random screening of large libraries of candidatesubstances; alternatively, the assays may be used to focus on particularclasses of compounds selected with an eye towards structural attributesthat are believed to make them more likely to modulate the expression ofa desired target protein or proteins.

To identify a modulator, one generally will determine protein expressionin the presence and absence of the candidate substance, a modulatordefined as any substance that alters the expression. For example, amethod generally comprises:

-   -   (a) providing a candidate modulator;    -   (b) contacting the candidate modulator with a tissue sample;    -   (c) measuring protein content in the tissue sample of step (b);        and    -   (d) comparing the content in step (c) with the content observed        in the absence of the candidate modulator,    -   wherein a difference between the measured characteristics        indicates that said candidate modulator is, indeed, a modulator        of the compound, cell or animal.        It will, of course, be understood that all the screening methods        of the present invention are useful in themselves        notwithstanding the fact that effective candidates may not be        found. The invention provides methods for screening for such        candidates, not solely methods of finding them. As stated above,        the invention may also be applied to the examination of nucleic        acids, lipids, carbohydrates, or metabolites.

1. Modulators

As used herein the term “candidate modulator” refers to any moleculethat may alter stability or expression of a protein target or set ofprotein targets. The candidate substance may itself be a protein orfragment thereof, a small molecule, or even a nucleic acid molecule.Using lead compounds to help develop improved compounds is know as“rational drug design” and includes comparisons with know inhibitors andactivators. The goal of rational drug design is to produce structuralanalogs of biologically active modulators. By creating such analogs, itis possible to fashion drugs, which are more active or stable than thenatural molecules, which have different and desirable properties.

On the other hand, one may simply acquire, from various commercialsources, small molecule libraries that are believed to meet the basiccriteria for useful drugs in an effort to “brute force” theidentification of useful compounds. Screening of such libraries,including combinatorially generated libraries (e.g., peptide libraries),is a rapid and efficient way to screen large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third and fourth generation compounds modeled of active, butotherwise undesirable compounds.

Candidate compounds may include fragments or parts ofnaturally-occurring compounds, or may be found as active combinations ofknown compounds, which are otherwise inactive. It is proposed thatcompounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived or synthesized from chemicalcompositions or man-made compounds. Thus, it is understood that thecandidate substance identified by the present invention may be peptide,polypeptide, polynucleotide, small molecule inhibitors or any othercompounds that may be designed through rational drug design startingfrom known inhibitors or stimulators.

Other suitable modulators include antisense molecules, ribozymes, andantibodies (including single-chain antibodies). Such compounds aredescribed in greater detail elsewhere in this document. For example, anantisense molecule that bound to a translational or transcriptionalstart site, or splice junctions, would be putative inhibitor of proteinexpression.

In addition to the modulating compounds initially identified, theinventors also contemplate that other sterically similar compounds maybe formulated to mimic the key portions of the structure of themodulators. Such compounds, which may include peptidomimetics of peptidemodulators, may be used in the same manner as the initial modulators.

2. In Situ Assays

In accordance with the present invention, examination of discreteregions of a treated tissue sample may be accomplished. In someembodiments, the tissue as a whole may be treated, either before orafter its obtention from an organism. In other embodiments, theindividual microwells or regions of the tissue may be treated aftersample obtention and preparation. In whole tissue treatments, a benefitwill be derived from the ability to perform replicate samples in ahomogenous tissue environment. Treatment of subregions followingobtention provides the advantage that treatments may be varied acrossdifferent wells or subregions with the expectation that the underlyingmaterial is of equivalent quality and does not vary substantially in itspretreatment state.

D. Assessing Delivery of an Agent

In a distinct embodiment from those discussed above, tissue microregionsare examined not for expression of endogenous proteins, but for anexogenous agent that is delivered to the tissue. The endogenous agentmay be a protein, a peptide (natural or synthetic), a nucleic acid (DNA,RNA, expression constructs, oligonucleotides, antisense, ribozymes,siRNA), or small molecule organopharmaceuticals.

The assessment may be designed to determine the ability of the agent tobe delivered to and persist in a target tissue. In addition, it may bedesirable to assess the ability of the agent to create anothernon-endogenous product, such as a protein expressed from an expressionvector, a drug created from a prodrug, or a product created from anenzyme.

VI. Examples

The following examples are included to further illustrate variousaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques and/or compositions discovered by the inventor tofunction well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Example 1 Scanning Electron Microscopy (SEM) Analysis of the Structureof Microwells in Rat Liver Tissue

Microwells in rat liver tissue were produced by the following method.Rat liver tissue was cut into 12 μm thick sections and thaw mounted to agold coated MALDI target which was cut in 4 pieces. The tissue waswashed 2× for 30 sec in 70% ethanol followed by a 15 sec wash in 100%ethanol. The section was then stored in a vacuum desiccator for 10minutes. Holes were drilled using the RapidSpotter using the followingconditions: start stop mode, 6×5 DPS at a 10 Hz drop ejection rate. Thesolvent was a mixture of 30% isopropanol, 10% acetonitrile and water towhich 0.1% acetic acid was added.

The tissue was stored for 4 days in desiccator, then analyzed byscanning electron microscopy (SEM). A thin layer of a metal wassputtered onto the section immediately prior to the analysis. Tissue wasanalyzed on a CM-12 Electron microscope from Hitachi. Accelerationvoltage was 15 kV and filament current was 10 μA. SEM Images werecaptured and saved to disc. Adobe Photoshop was used for further imageprocessing which included contras and brightness adjustments.

To investigate the three dimensional structure of a well, a tiltexperiment was performed. The sample stage of the SEM instrument wastilted from −3 up to +9 degrees and images were obtained.

FIG. 1 shows an image of microwells in the rat liver tissue which wassubsequently analyzed by SEM. FIG. 2A and FIG. 2B show SEM images of themicrowells, and these results indicate that the well evaluated does notreach the bottom of the plate on which the tissue was fixed upon. Theseresults are supported by FIG. 3A, FIG. 3B, and FIG. 3C. SEM imagesshowing images taken from different angles are shown in FIG. 4A, FIG.4B, FIG. 4C, and FIG. 4D. These results indicate that the microwellstructure is particularly well suited for in situ analysis.

Example 2 In Situ Analysis of Microwells

Several aspects of in situ analysis of microwells are evaluated in thisexample, including: performing in situ reactions in or on tissue,investigating well structures as observed when solvent is applied to thesurface of tissue, and performing protein quantification and expressionanalysis using chemical printing.

In situ MALDI mass spectroscopy was evaluated on microwells in rat livertissue. Microwells were generated in rat liver tissue by dispensing asolution on the tissue using the RapidSpotter. FIG. 5 shows spotting ofspecial matrix solution onto washed rat liver tissue. The tissue waswashed 2× with 70% Ethanol (30 s each) and 1×15 sec with 100% ethanol.After 2 DPS of a special matrix mix, tissue was gone (FIG. 5). Thematrix mix was: saturated solution of sinapinic acid in 30% isopropanol,10% acetonitrile, 60% H₂O and 0.5% acetic acid. No signals from holeswas detected, and the solubility of the matrix was very low. It wasobserved that the tissue changed aspect during 100% ethanol wash. Theresults indicate that the tissue dehydrated very well. MALDI spectraobtained after respotting of microwells with 20 mg/ml SA in 50%acetonitrile, 0.1% TFA was extremely good.

Microwells were also generated on mouse brain tissue. A 12 μm slice of amouse brain was ethanol washed with 70% ethanol (2×30 sec) and 100%ethanol (1×15 sec), and the tissue was spotted with 2×3DPS start stop 10Hz of a solvent (90% acetonitrile, 0.1% TFA). The results of the spotarray generated on the mouse brain tissue is shown in FIG. 6. Thisprocedure of drilling holes into a brain tissue increased the placementaccuracy if matrix was dispensed on top of the holes. Interestingly, themicrowells form on different regions of the tissue.

Microwells were generated on different tissues from various species.Microwells were generated on mouse uterus. A 12 μm slice of mouse embryotissue was ethanol washed with 70% ethanol (2×30 sec) and 100% ethanol(1×15 sec), and the tissue was spotted with 4×2 DPS solvent (90%acetonitrile, 0.1% TFA). These results in the mouse uterus tissue areshown in FIG. 7A. Microwells were also generated on human brain tissue.A 12 μm slice of human brain tissue was spotted with solvent (90%acetonitrile, 0.1% TFA), and the results are indicated in FIG. 7B.

Microwells were also generated on rat kidney tissue. A 12 μm slice ofrat kidney was spotted using 1×, 2× and 3×3 DPS 90% acetonitrile, 0.1%TFA. The results of these experiments is shown in FIG. 8A, FIG. 8B, FIG.8C, and FIG. 8D. These results indicate a good placement accuracy of thematrix which was spotted on the tissue.

Confocal microscopy was used to evaluate the structure of microwells inmouse brain tissue. A 25 μm thick slice of Mouse brain tissue wasethanol washed. Microwells were spotted with 10×3DPS of 90%acetonitrile, 0.1% TFA. Confocal microscopy images are shown in FIGS.9A-H. The mouse brain tissue was DIO stained. The laser used was 488 nm.Fluorescence was detected above 505 nm.

Optical microscopy was performed to evaluate a microwell structureproduced in rat liver tissue. Solvent was spotted onto rat liver tissueand optical microscopy images are shown in FIGS. 10A-B. 12 μm slices ofrat liver tissue on conductive glass slides are shown. Images wereobtained using a microscope 40× magnification. These results indicatethat the membrane in this region was completely dissolved.

Mass spectroscopy (MS) was performed on a microwell in adult mouse braintissue. An adult mouse brain was first sectioned on the cryostat (12 μmthickness). The tissue was washed in two consecutive baths of 70%ethanol for 30 sec and 15 sec in 100% ethanol. The washed tissue sectionwas stored in a vacuum desiccator for 1 h. 500 μl of a 10 mmol/lammonium carbonate buffer solution (pH 8.2, 20° C.) was manuallypipetted onto the tissue followed by 500 nl of sequencing grade trypsinprepared at 5 pmol/μl in 2 mmol/l HCl solution. The tissue was thenincubated for 30 min at 39° C. in a humidified chamber. The tissue wasnext allowed to dry on the bench. 500 μl of matrix solution consistingof 10 mg/ml CHCA and 1 mg/ml ammonium citrate (dibasic) in 50%acetonitrile 0.1% TFA was spotted onto each reaction site. Spectraacquisition was obtained with the 4700 proteomics analyzer from AppliedBiosystems. FIGS. 11A-C show results indicating the identification ofthe mouse β tubulin protein Q9CWF2. FIGS. 12A-B indicate theidentification of the myelin basic protein. External calibrations wereperformed using peptide standard mixtures on plates.

Example 3 Ethanol Pretreatment

In order to assess the impact of pretreatments of microwell formation,ethanol washes were performed on cell sections prior to creation ofmicrowells. Tissues were 12 μm thick rat liver on conductive glassslides. The tissues were washed 2× for 30 secs in 70% ethanol followedby 1 wash for 15 secs in reagent grade ethanol. The tissue was storedovernight in a dessicator. Good well formation was observed.

In a second experiment, three different pretreatments were performed onrat liver sections: (a) ethanol wash followed by 130 min in vacuumdessicator; (b) ethanol wash followed by 22 min in vacuum dessicator;(c) no wash followed by 180 min in vacuum dessicator. Again, the ethanolwashed tissue gave better results, as did the longer storage samples.

A third experiment using mouse brain tissue, again 12 μm in thickness,was performed. The cryosectioned tissue was thaw mounted on a conductiveglass slide and either washed in ethanol or not washed. Solvent (90%acetonitrile containing 0.1% TFA) was spotted at 10 Hz ejectionfrequency. While holes were produced in both tissues, ethanol washingimproved hole formation. There was no visible difference between holesformed at 3×3 and 3×6 DPS.

Example 4 Preparation of Wells Using Solvents and Seeding

In order to assess the performance of various solvents, five differentformulations were tested: (a) 30% isopropanol, 10% acetonitrile, 0.5%acetic acid, 60% water; (b) 20% water, 20% acetic acid, 60% isopropanol;(c) 50% acetonitrile, 0.1% TFA; (d) water; and (e) 860 μm Triton x-100(CMC is 300 μM), 0.15 M NaCl, in 50 mM Tris/HCl buffer pH=7.45 (20° C.).Solvent 1 improved MALDI signals in the low molecular weight range,Solvent 2 seemed to lyse efficiently but the MALDI spectrum showedmainly hemoglobin and Solvent 5 also was dominated by hemoglobin peaks.

Another approach to preparing wells involves use of a seeding materialto initiate and homogenize crystal formation. Ethanol washed rat liverwas spotted with 200 nl of a saturated sinapinic acid solution in asolvent mixture (10% acetonitrile, 60% water, 30% isopropanol, 0.5%acetic acid) followed by 200 nl of sinapinic acid prepared at 25 mg/mlin 50% acetonitrile containing 0.1% TFA. MALDI crystals were visible inthe well.

Example 5 Number of Droplets Versus Number of Print Passes

To assess the impact of multiple droplets on well formation, thefollowing experiment was set up on 12 μm rat liver sections. Ethanolwashed tissue was spotted with 1-9 droplets (start/stop mode) of thefollowing solvent: 0.5% acetic acid mixed to a solvent mixture of 30%isopropanol, 10% acetonitrile and 60% water. Droplets deposited A) 1DPS, B) 2 DPS, C) 3 DPS, D) 4DPS, E) 5 DPS, F) 6 DPS, G) 7 DPS, H) 8DPS, I) 9 DPS. As show in FIG. 13, the more droplets used, the morepronounced the wells.

To assess the effects of multiple print passes, rat liver 12 μm wasassessed using 4, 6 or 10 passes (A is 4 times, B is 6 times, C is 10times) and 1) 3 DPS start stop, 2) 5 DPS start stop, or 3) 10 DPS startstop. The solvent was 0.5% acetic acid mixed to a solvent mixture of 30%isopropanol, 10% acetonitrile and 60% water. As show in FIGS. 14A-B,again, more droplets give better wells, and the wells increase in sizewith passes. FIG. 14C shows a close-up of a stained well.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods, and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

VIII. References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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1. A method for analyzing protein content in a tissue comprising: (a)providing an intact tissue comprising a first spatially discretemicrowell; (b) subjecting said microwell to one or more physical orchemical treatments; and then (c) analyzing a protein sample from saidmicrowell with secondary ion mass spectrometry, laser desorption massspectrometry, matrix-assisted laser desorption mass spectrometry,desorption electrospray or electrospray mass spectrometry, therebyproviding analysis of protein content in said tissue.
 2. The method ofclaim 1, wherein said intact tissue comprises at least a secondspatially discrete microwell, and said method further comprisessubjecting said second spatially discrete microwell to one or morephysical or chemical treatments, and analyzing discrete protein samplesfrom said second spatially discrete microwell.
 3. The method of claim 2,wherein the protein content of said first and second spatially discretemicrowells are compared.
 4. The method of claim 1, wherein said tissueis an animal tissue.
 5. The method of claim 4, wherein said animaltissue is heart tissue, liver tissue, kidney tissue, prostate tissue,breast tissue, ovary tissue, uterine tissue, skin tissue, lung tissue,brain tissue, colon tissue, head & neck tissue, pancreatic tissue,muscle tissue or skin or tissue that contains body fluids or containstraces of such fluids.
 6. The method of claim 4, wherein said animaltissue is diseased or injured.
 7. The method of claim 6, wherein saiddiseased tissue is cancerous, inflamed, infected, congenitally diseasedor functionally compromised.
 8. The method of claim 6, wherein saidinjured tissue is traumatized or environmentally insulted.
 9. The methodof claim 1, wherein said tissue is plant tissue.
 10. The method of claim9, wherein said plant issue is leaf tissue, stalk tissue, stem tissue,root tissue, or seed tissue.
 11. The method of claim 9, wherein saidplant tissue is diseased or injured.
 12. The method of claim 11, whereinsaid diseased tissue is infected, or congenitally diseased.
 13. Themethod of claim 11, wherein said injured tissue is traumatized orenvironmentally insulted.
 14. The method of claim 1, wherein said one ormore physical or chemical treatments comprise solvent treatment,detergent treatment, lipase treatment, proteolysis, reactive agenttreatment or labeling.
 15. The method of claim 14, wherein labelingcomprises treatment with an isotope dilution reagent, a labeledantibody, or an enzyme.
 16. The method of claim 1, wherein step (c) isperformed in situ in said microwell.
 17. The method of claim 1, whereinstep (c) is performed after removing said protein sample from saidmicrowell.
 18. The method of claim 2, wherein step (c) is performed insitu in said spatially discrete microwells.
 19. The method of claim 2,wherein said first and second spatially discrete microwells receivedistinct physical or chemical treatments.
 20. The method of claim 2,wherein said first and second spatially discrete microwells receive thesame physical or chemical treatment.
 21. The method of claim 2, whereinsaid first spatially discrete microwell or tissue adjacent to said firstspatially microwell is subject to a first test condition prior to step(b).
 22. The method of claim 21, wherein said first test conditioncomprises a drug treatment, a nutrient treatment, a hormone treatment,an enzyme treatment, or a cytokine treatment.
 23. The method of claim21, further subjecting said first spatially discrete microwell or tissueadjacent to a said first spatially discrete microwell to a second testcondition prior to step (b).
 24. The method of claim 21, wherein saidsecond test condition is different from said first test condition. 25.The method of claim 21, wherein said second test condition is the sameas said first test condition.
 26. The method of claim 1, wherein saidfirst spatially discrete microwell is subjected to at least a secondphysical or chemical treatment.
 27. The method of claim 26, wherein saidsecond physical or chemical treatment is distinct from said firsttreatment.
 28. The method of claim 1, wherein said microwell is between5 and 200 microns in diameter.
 29. The method of claim 1, wherein saidmicrowell is between 10 and 100 microns in diameter.
 30. The method ofclaim 1, wherein said microwell is about 50 microns in diameter.
 31. Themethod of claim 1, further comprising generating said microwell.
 32. Themethod of claim 31, further comprising generating a plurality ofmicrowells or a microwell array.
 33. The method of claim 2, wherein saidintact tissue comprises 6, 24 or 96 microwells.
 34. The method of claim1, wherein one or both of steps (b) and (c) are automated.
 35. A methodfor analyzing an endogenous metabolite in a tissue comprising: (a)providing an intact tissue comprising a first spatially discretemicrowell; (b) subjecting said microwell to one or more physical orchemical treatments; and then (c) analyzing an endogenous metabolitesample from said microwell by secondary ion mass spectrometry, laserdesorption mass spectrometry, matrix-assisted laser desorption massspectrometry, desorption electrospray or electrospray mass spectrometry,thereby providing analysis of endogenous metabolite content in saidtissue.
 36. The method of claim 35, wherein said endogenous metaboliteis an organic acid metabolite, peptide metabolites, or a part of asugar.